Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL COMPOSITION WITH BISPHOSPHONATE
Field of the Invention
The present invention relates to depot formulations comprising a poorly water
soluble
salt (also referred to as poorly soluble salt hereinafter, meaning poorly
water soluble) of a
bisphosphonate forming together with one or more biocompatible polymers. The
depot
formulation may be in the form of microparticles or implants. The depot
formulations are
useful for the treatment and prevention of various, e.g. bone related and/or
proliferative,
diseases, especially degenerative diseases and rheumatoid arthritis and
osteoarthritis.
In a further aspect, the present invention relates to new salts including new
crystal
forms of said salts of certain bisphosphonates, as well as new crystal forms
of the
bisphosphonates in free (e.g. zwitterionic) form.
Further, various other embodiments (uses, methods, processes or methods for
preparation and related subject matter) are embodiments of the invention.
Background of the Invention
Bisphosphonates are widely used to inhibit osteoclast activity in a variety of
both
benign and malignant diseases in which bone resorption is increased. So far,
only water
soluble bisphosphonates, e.g., the sodium salt, have been used in
pharmaceutical
compositions. In case of forming solutions for infusion this is a reasonable
approach.
However, in case of a depot formulation the high water solubility of the
bisphosphanate will
lead to a high initial release causing severe local tissue irritations.
For example, the drug zoledronic acid is used in the prevention of skeleton
related
events, such as, inter alia, pathological fractures, spinal compression,
radiation or surgery to
bone or tumor-induced hypercalcemia) in patients with various diseases or
disorders e.g.
involving bone and calcium metabolism, such as advanced malignancies involving
bone,
treatment of tumor-induced hypercalcemia, Paget's disease, operation and
prevention of hip
fractures, or the like.
Overview over the Invention
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It has now been surprisingly found that poorly water soluble bisphosphonates
of a
novel class of bisphosphonates can be encapsulated very efficiently so that
the drug release
is very well under control.
One advantage of a poorly water soluble salt is that generally the
encapsulation of the drug
substance is improved because highly water soluble salts may dissolve into the
aqueous
phase during the manufacturing of the microparticles via commonly used
emulsion-solvent
evaporation/extraction method. A further advantage is that the drug release
out of the
resulting depot formulation is generally better controlled if the drug
substance has limited
water solubility compared to highly water soluble salts.
An advantage of the micronization of the drug substance is the more complete
encapsulation
of the drug substance particles in polymer matrices compared to large drug
substance
particles which may only partly been encapsulated in the matrix leading to an
uncontrolled
release of the drug substance.
In addition, new salts have been found that enable the manufacture of the
aforementioned
formulations.
Further, new crystal forms of certain bisphosphonates and their salts
(including hydrates or
other solvates) have been found and are an embodiment of the invention.
Detailed Description of the Drawings
Fig. 1 shows the X-ray diffractogram of the crystalline zwitterionic
(internal) salt of [2-(5-
ethyl-imidazol-1-yl)-1-hydroxy-1-phosphono-ethyl]-phosphonic acid (Cpd. A),
for details see
Example 6.
Fig. 2 shows the X-ray diffractogram of the crystalline Ca-salt of Cpd. A
(1:2), for details see
Example 7.
Fig. 3 shows the X-ray diffractogram of the crystalline Mg-salt of Cpd. A
(1:2), for details see
Example 8.
Fig. 4 shows the X-ray diffractogram of the crystalline Zn-salt of Cpd. A
(1:2), for details see
Example 9.
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Detailed Description of the Invention
In the following detailed description of the invention, more specific
definitions provided for
general terms in one of the embodiments may also be used to define the general
terms
more specifically in other embodiment, this forming more specific embodiments
of the
invention, with the proviso, that each term may be replaced independently of
the other
general terms defining an embodiment of the invention.
The present invention, in a first embodiment, relates to depot formulations
comprising a
poorly water soluble salt of a bisphosphonate of the formula I together with
biocompatible
polymers.
The present invention in this regard especially relates to depot formulations
comprising a
poorly water soluble salt of a bisphosphonate forming together with one or
more
biocompatible polymers, where the bisphosphonate compound is a compound
selected from
compounds of the formula I,
OHOH
R1 o`
OH
R2 N
0P
/
OH
HO
(I)
wherein one of R, and R2 is hydrogen and the other is C,-C5-alkyl (preferably
C2-C5alkyl) that
is branched or unbranched in the form of a poorly water-soluble salt.
Preferred is a depot formulation of the bisphosphonate of the formula I,
wherein one
of R, and R2 is hydrogen and the other is methyl in the form of a poorly water-
soluble salt.
Alternatively, a depot formulation of the bisphosphonate of the formula I
wherein one of R,
and R2 is hydrogen and the other is ethyl in the form of a poorly water-
soluble salt is very
preferred.
Most preferred is a depot formulation of the bisphosphonate of the formula I
with the
name [2-(5-methyl-imidazol-1-yl)-1-hydroxy-1-phosphono-ethyl]-phosphonic acid
or more
preferably [2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1-phosphono-ethyl]-phosphoric
acid in the
form of a poorly water-soluble salt.
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Also the poorly water-soluble salts of compounds of the formula I, especially
the salts
with the preferred compounds of the formula I as defined in the preceding
paragraphs, as
such are an embodiment of the invention, especially in the form of specific
polymorphs
(crystal forms or crystal modifications) as described below in more detail.
"Poorly soluble", wherever used in this text, means that the solubility is 2
mg/ml in
water at a temperature from 21 to 24 C, more preferably less than 1 mg/ml of
water at said
temperature.
The present invention relates especially to depot formulations in the form of
microparticles
comprising a poorly water soluble salt of a bisphosphonate of the formula I
together with one
or preferably more biocompatible polymers, preferably biodegradable polymers.
The present invention also relates to implants comprising a poorly water
soluble salt of a
bisphosphonate of the formula I together with one or preferably more
biocompatible
polymers, preferably biodegradable polymers.
The present invention relates to methods for the treatment and prevention of
diseases or
disorders where abnormal bone turnover is found, as provided in more detail
below,
comprising administering a depot formulation or a poorly soluble salt or a
crystalline form of
a free form (or its internal salt, e.g. zwitterionic salt) of a compound of
the formula Ito a
patient in need of such treatment in a therapeutically effective dosage, as
well as the use of
a depot formulation or poorly soluble salt or a crystalline form of a free
form (or its internal
salt, e.g. zwitterionic salt) of a compound of the formula I in the
manufacture of medicaments
for the treatment of such diseases or disorders and their use in the treatment
of said
disorders or diseases, as well as the depot formulations or salts a
crystalline forms of a free
form (or its internal salt, e.g. zwitterionic salt) of a compound of the
formula I for use in such
treatment.
The poorly water-soluble salt of a compound of the formula I which is an
embodiment of the
invention or is part of a depot formulation according to the invention is
selected from the
calcium, magnesium and zinc salt, or a mixture of two or all of these salts,
preferably as 1:1
or especially 1:2 salts (herein wherever mentioned giving the molar ratio of
(metal ion) :
(compound of the formula I), where "metal" refers to calcium, magnesium and/or
(especially
"or") zinc). These salts are low in water solubility, in other terms, poorly
water soluble means
that the water solubility is 25 % or less of a corresponding sodium salt.
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Preferably, the depot formulations of the invention contain as active
ingredient only a
compound of formula I, preferably [2-(5-methyl-imidazol-1-yl)-1-hydroxy-1-
phosphono-ethyl]-
phosphonic acid or especially [2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1-phosphono-
ethyl]-
phosphonic acid, in the form of its poorly water soluble salt, or a
crystalline form of a
compound of the formula I named [2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1-
phosphono-ethyl]-
phosphonic acid in free (e.g. especially zwitterionic) form.
It has been found that the calcium salts are better polymer-encapsulated in
the formulations
according to the invention than the zinc salts - therefore, calcium salts of a
compound of the
formula I are generally more preferred, especially for the depot formulations.
In addition, the defined crystal forms both of the free compounds as well as
the salts of the
compounds of the formula I, respectively, show additional advantages, e.g. a
fixed
stochiometric relationship between their components and, where solvates, such
as hydrates,
are formed, the solvent molecules, good millability to yield particles in the
micrometer range,
good flowability and other advantageous properties of crystalline over
amorphic materials
that facilitate the processing of such materials to pharmaceutical
formulations, also including
improved storability.
Preferably, the microparticles of the invention contain a compound of formula
I, in form of the
calcium salt, even more preferably the calcium salt of [2-(5-ethyl-imidazol-1-
yl)-1-hydroxy-1-
phosphono-ethyl]-phosphonic acid.
The bisphosphonates of the formula I may be present in an amount of from about
1 % to
about 60%, more usually about 2% to about 20%, preferably about 5% to about
10%, by
weight of the depot dry weight of the microparticle formulation.
The bisphosphonates of the invention are released from the depot formulations
of the'
invention and from the compositions of the invention over a period of several
weeks, e.g.,
about 2 weeks to 18 months, e.g. from 3 weeks to 12 months.
Preferably, the bisphosphonate of the formula I in the form of its poorly
water-soluble salt
used to prepare the depot formulations is a very fine powder produced by any
type of micro-
nization technique (e.g., jet milling or high pressure homogenization) having
a particle size
(e.g. with 90 % of the weight of the particles in that range, preferably 98 %)
of about 0.1
microns to about 15 microns, preferably less than about 5 microns, even more
preferably
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less than about 3 microns. It is found that micronizing the drug substance
improves the
encapsulation efficiency.
In accordance with one aspect, the invention a calcium salt of a compound of
the formula I,
especially with a stoichiometry of one calcium and two molecules of compound
of the
formula I (an 1:2 salt), especially a calcium salt of Compound A (= Cpd. A =
[2-(5-ethyl-
imidazol-1-yl)-1-hydroxy-1-phosphono-ethyl]-phosphonic acid).
In accordance with another aspect, the invention provides a zinc salt of a
compound of the
formula I, especially with a stoichiometry of one zinc and two molecules of
compound of the
formula I (an 1:2 salt) or of one zinc and two molecules of compound of the
formula I (an 1:1
salt), especially a zinc salt of Compound A (= Cpd. A = [2-(5-ethyl-imidazol-1-
yl)-1-hydroxy-
1-phosphono-ethyl]-phosphonic acid).
In accordance with yet another aspect, the invention provides a magnesium salt
of a
compound of the formula I, especially with a stoichiometry of one Magnesium
and two
molecules of compound of the formula I (a 1:2 salt), especially a magnesium
salt of
Compound A (= Cpd. A = [2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1-phosphono-ethyl]-
phosphonic acid).
Moreover, it has surprisingly been found that the compounds of the formula I
in free form
(this term always including internal salts, such as zwitterionic forms) as
well as salts of
compounds of the formula I can be present in polymorphic forms (different
crystal
modifications).
The invention therefore in a further embodiment relates to new crystalline
forms of low water
soluble salts of compounds of the formula I or their free (e.g. zwitterionic)
form, especially of
[2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1-phosphono-ethyl]-phosphonic acid (Cpd.
A
hereinafter), the process for preparation of these crystalline forms,
compositions containing
these crystalline forms, and the use of these crystalline forms in diagnostic
methods or
therapeutic treatment of warm-blooded animals, especially humans.
Both the free forms as well as the salt forms, each in crystalline form, may
be free of solvent
or (especially in the case of the salts) in solvate, e.g. hydrate form, e.g.
as the dihydrate.
With regard to the crystalline forms, the invention, in a first aspect,
provides a crystalline
form of the free form or one of the salt forms (especially a salt in hydrate
form) of a
compound of the formula I.
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In a more focused aspect, the invention provides a crystalline form of the
free zwitterionic
form of Cpd. A, which more preferably has an X-ray powder diffraction pattern
with at least
one, preferably two, more preferably three, most preferably all of the
following peaks at an
angle of refraction 2 theta (8) of 10.5, 13.1, 14.7, 17.2, 23.5, 25.2 and
29.2, 0.2,
respectively, especially as depicted in Figure 1; alternatively, at least 80 %
by weight of Cpd.
A in the free zwitterionic form shows such X-ray powder diffraction pattern.
In another more focused aspect, the invention provides a crystalline form of
the calcium salt
of Cpd. A (especially in the hydrate form, such as the dihydrate) with a
stoichiometry of one
calcium and two molecules of Cpd. A, which more preferably has an X-ray powder
diffraction
pattern with at least one, preferably two, more preferably three, most
preferably all of the
following peaks at an angle of refraction 2 theta (8) of 7.9, 10.6, 12.1,
25.7, 27.4 and 29.2,
0.2, respectively, especially as depicted in Figure 2; alternatively, at least
80 % by weight of
the calcium 1:2 salt of Cpd. A shows such X-ray powder diffraction pattern.
In yet another more focused aspect, the invention provides a crystalline form
of the zinc salt
of Cpd. A (especially in the hydrate form, such as the dihydrate) with a
stoichiometry of one
zinc and two molecules of Cpd. A, which more preferably has an X-ray powder
diffraction
pattern with at least one, preferably two, more preferably three, most
preferably all of the
following peaks at an angle of refraction 2 theta (8) of 6.7, 9.5, 12.5, 17.7
and 27.3, 0.2,
respectively, especially as depicted in Figure 3; alternatively, at least 80 %
by weight of the
zinc 1:2 salt of Cpd. A shows such X-ray powder diffraction pattern.
In yet another more focused aspect, the invention provides a crystalline form
of the magne-
sium salt of Cpd. A (especially in the hydrate form, such as the dihydrate)
with a stoichio-
metry of one magnesium and two molecules of Cpd. A, which more preferably has
an X-ray
powder diffraction pattern with at least one, preferably two, more preferably
three, most
preferably all of the following peaks at an angle of refraction 2 theta (6) of
6.7, 12.5, 20.0
and 27.3, 0.2, respectively, especially as depicted in Figure 4;
alternatively, at least 80 %
by weight of the magnesium 1:2 salt of Cpd. A shows such X-ray powder
diffraction pattern.
The parameters and devices for retrieval of the X-ray data mentioned above and
in the
claims preferably are in accordance with those mentioned below in the
Examples.
In accordance with another aspect of the invention, the invention provides a
pharmaceutical
formulation (especially a depot formulation as herein described) including a
crystalline form,
especially as described in any one of the above.mentioned focused aspects of
the invention,
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of a compound of the formula I or a poorly soluble salt thereof, especially a
calcium salt
(calcium : Cpd. A = 1 : 2 being especially preferred, especially in the
hydrate, e.g. dihydrate,
form) and at least one pharmaceutically acceptable carrier, especially for
parenteral
administration.
In yet another aspect, the invention relates to an amorphous or crystalline
form of a
compound of the formula I, especially Cpd. A, in the form of a poorly soluble
salt selected
from the zinc, (especially) magnesium and (more especially) calcium salt,
especially where
the stoichiometry of the metal ion to the compound of the formula I is 1:2; or
to a crystalline
form of a compound of the formula I, especially in its free (e.g. inner
zwitterionic) form or in
the form of an (especially 1:1 or more especially 1:2) zinc, (especially)
magnesium or (more
especially) calcium salt, each especially in hydrate form, e.g. in the form of
a dihydrate, or a
mixture of two or more such forms, especially for use in the treatment of one
or more
diseases or disorders where abnormal bone turnover is found (the term
treatment wherever
used in this disclosure including both prophylactic and therapeutic (e.g.
palliative or curing)
treatment.
About, where used in this specification, especially means that the number
mentioned after
"about" can vary by plus 10 to minus 10 percent of its absolute value.
The particle size distribution of the poorly water-soluble salts of
bisphosphonates of the
formula I may influence the release profile of the drug. Typically, the
smaller the particle
size, the lower is the burst and release during the first diffusion phase,
e.g., the first 20 days.
Preferably, particle size distribution is, e.g., x 10 < 2 microns, i.e., 10%
of the particles are
smaller than 2 microns; x 50 < 5 microns, i.e., 50% of the particles are
smaller than 5
microns; or x 90 < 10 microns, i.e., 90% of the particles are smaller than 10
microns.
11. Microparticles
It has been found that administration of microparticles comprising a low
soluble salt
of a bisphosphonate of the formula I embedded in a biocompatible
pharmacologically
acceptable polymer, preferably a biodegradable pharmacologically acceptable
polymer,
suspended in a suitable vehicle gives release of the active agent over an
extended period of
time, e.g., one week up to 18 months, preferably for about 3 weeks to about 12
months.
The present invention in another aspect provides a process for the preparation
of
microparticles of the invention comprising:
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(i) preparation of an internal organic phase comprising:
(ia) dissolving the polymer or polymers in a suitable organic solvent or
solvent
mixture, and optionally dissolving/dispersing a porosity-influencing agent in
the
solution obtained in step (ia), or
- adding a basic salt to the solution obtained in step (ia),
- adding a surfactant to the solution obtained by step (ia);
(ib) suspending a poorly water-soluble salt of a compound of the formula I in
the
polymer solution obtained in step (ia), or dissolving a poorly water-soluble
salt of
a compound of the formula I in a solvent miscible with the solvent used in
step
(ia) and mixing said solution with the polymer solution, or directly
dissolving a
poorly water-soluble salt of a compound of the formula I in the polymer
solution;
(ii) preparation of an external aqueous phase comprising
(iia) preparing a buffer to adjust the pH to 3.0-8.0, for example pH 3.0-5.0;
e.g.,
acetate buffer, and
(iib) dissolving a stabilizer in the solution obtained in step (iia);
(iii) mixing the internal organic phase with the external aqueous phase, e.g.,
with a
device creating high shear forces, e.g., with a turbine or static mixer, to
form an
emulsion; and
(iv) hardening the microparticles by solvent evaporation or solvent
extraction,
optionally in addition washing the microparticles, e.g., with water, and
collecting and
drying the microparticles, e.g., by freeze-drying or drying under vacuum.
Suitable organic solvents for the polymers include, e.g., ethyl acetate or
halogenated
hydrocarbons, e.g., methylene chloride, chloroform, or mixtures of two or more
e.g. of them.
Suitable examples of a stabilizer for step (iib) include:
a) Polyvinyl alcohol (PVA), preferably having a weight average molecular
weight from
about 10,000 Da to about 150,000 Da, e.g., about 30,000 Da. Conveniently, the
polyvinyl alcohol has low viscosity having a dynamic viscosity of from about 3
mPa s to
about 9 mPa s when measured as a 4% aqueous solution at 20 C or by DIN 53015.
Suitably, the polyvinyl alcohol may be obtained from hydrolyzing polyvinyl
acetate.
Preferably, the content of the polyvinyl acetate is from about 10% to about
90% of the
polyvinyl alcohol. Conveniently, the degree of hydrolysis is about 85% to
about 89%.
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Typically the residual acetyl content is about 10-12%. Preferred brands
include
Mowiol 4-88, 8-88 and 18-88 available from Kuraray Specialities Europe, GmbH.
Preferably the polyvinyl alcohol is present in an amount of from about 0.1 %
to about
5%, e.g., about 0.5%, by weight of the volume of the external aqueous phase;
b) Hydroxyethyl cellulose (HEC) and/or hydroxypropyl cellulose (HPC), e.g.,
formed by
reaction of cellulose with ethylene oxide and propylene oxide, respectively.
HEC and
HPC are available in a wide range of viscosity types; preferably the viscosity
is
medium. Preferred brands include Natrosol from Hercules Inc., e.g., Natrosol
250MR and Klucel from Hercules Inc.
Preferably, HEC and/or HPC is present in a total amount of from about 0.01% to
about
5%, e.g., about 0.5%, by weight of the volume of the external aqueous phase;
c) Polyvinylpyrolidone, e.g., suitably with a molecular weight of between
about 2,000 Da
and 20,000 Da. Suitable examples include those commonly known as Povidone K12
F
with an average molecular weight of about 2,500 Da, Povidone K15 with an
average
molecular weight of about 8,000 Da, or Povidone K17 with an average molecular
weight of about 10,000 Da. Preferably, the polyvinylpyrolidone is present in
an amount
of from about 0.1% to about 50%, e.g., 10% by weight of the volume of the
external
aqueous phase
d) Gelatin, preferably porcine or fish gelatin. Conveniently, the gelatin has
a viscosity of
about 25 cps to about 35 cps for a 10% solution at 20 C. Typically pH of a 10%
solution is from about 6 to about 7. A suitable brand has a high molecular
weight, e.g.,
Norland high molecular weight fish gelatin obtainable from Norland Products
Inc,
Cranbury, New Jersey, USA.
Preferably, the gelatin is present in an amount of from about 0.01 % to about
5%, e.g.,
about 0.5%, by weight of the volume of the external aqueous phase.
Preferably, polyvinyl alcohol is used. Preferably, no gelatin is used.
Preferably, the
microparticles are gelatin-free.
The resulting microparticles may have a diameter from a few submicrons to a
few
millimeters; e.g., diameters of at most, e.g., 5-200 microns, preferably 5-130
microns, more
preferably 5-100 microns are strived for, e.g., in order to facilitate passage
through an
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injection needle. A narrow particle size distribution is preferred. For
example, the particle
size distribution may be, e.g., 10 % <20 microns, 50 % <50 microns or 90 % <80
microns.
Content uniformity of the microparticles and of a unit dose is excellent. Unit
doses
may be produced which vary from about 20% to about 125%, e.g., about 70% to
about
115%, e.g., from about 90% to about 110%, or from about 95% to about 105%, of
the
theoretical dose.
The microparticles in dry state may, e.g., be mixed, e.g., coated, with an
anti-
agglomerating agent, or, e.g., covered by a layer of an anti-agglomerating
agent, e.g., in a
prefilled syringe or vial.
Suitable anti-agglomerating agents include, e.g., mannitol, glucose, dextrose,
sucrose, sodium chloride or water soluble polymers, such as
polyvinylpyrrolidone or
polyethylene glycol, e.g., with the properties described above.
Preferably, an anti-agglomerating agent is present in an amount of about 0.1 %
to
about 10%, e.g., about 4% by weight of the microparticles.
Prior to (usually s.c. or i.m.) administration, the microparticles are
suspended in a
vehicle suitable for injection.
Accordingly, the present invention further provides a pharmaceutical
composition
comprising microparticles of the invention in a vehicle. The vehicle may
optionally further
contain:
a) one or more wetting agents; and/or
b) one or more tonicity agent; and/or
c) one or more viscosity increasing agents.
Preferably, the vehicle is water based, e.g., it may contain water, e.g.,
deionized, and
optionally a buffer to adjust the pH to 7-7,5, e.g., a phosphate buffer, such
as a mixture of
Na2HPO4 and KH2PO4, and one or more of agents a), b) and/or c) as indicated
above.
However, when using water as a vehicle, the microparticles of the invention
may not
suspend and may float on the top of the aqueous phase. In order to improve the
capacity of
the microparticles of the invention to be suspended in an aqueous medium, the
vehicle
preferably comprises a wetting agent a). The wetting agent is chosen to allow
a quick and
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suitable suspendibility of the microparticles in the vehicle. Preferably, the
microparticles are
quickly wettened by the vehicle and quickly form a suspension therein.
Suitable wetting agents for suspending the microparticles of the invention in
a water-
based vehicle include non-ionic surfactants, such as poloxamers, or
polyoxyethylene-
sorbitan-fatty acid esters, the characteristics of which have been described
above. A mixture
of wetting agents may be used. Preferably, the wetting agent comprises
Pluronic F68,
Tween 20 and/or Tween 80.
The wetting agent or agents may be present in about 0.01 % to about 1 % by
weight of
the composition to be administered, preferably from 0.01-0.5% and may be
present in about
0.01-5 mg/mL of the vehicle, e.g., about 2 mg/mL.
Preferably, the vehicle further comprises a tonicity agent b), such as
mannitol,
sodium chloride, glucose, dextrose, sucrose or glycerin. Preferably, the
tonicity agent is
mannitol.
The amount of tonicity agent is chosen to adjust the isotonicity of the
composition to
be administered. In case a tonicity agent is contained in the microparticles,
e.g., to reduce
agglomeration as mentioned above, the amount of tonicity agent is to be
understood as the
sum of both. For example, mannitol preferably may be from about 1% to about 5%
by
weight of the composition to be administered, preferably about 4.5%.
Preferably, the vehicle further comprises a viscosity increasing agent c).
Suitable
viscosity increasing agents include carboxymethyl cellulose sodium (CMC-Na),
sorbitol,
polyvinylpyrrolidone, or aluminum monostearate.
CMC-Na with a low viscosity may conveniently be used. Embodiments may be as
described above. Typically, a CMC-Na with a low molecular weight is used. The
viscosity
may be of from about 1 mPa s to about 30 mPa s, e.g., from about 10 mPa s to
about
15 mPa s when measured as a 1% (w/v) aqueous solution at 25 C in a Brookfield
LVT
viscometer with a spindle 1 at 60 rpm, or a viscosity of 1-15 mPa*s for a
solution of NaCMC
7LF (low molecular weight) as a 0.1-1 % solution in water.
A polyvinylpyrrolidone having properties as described above may be used.
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A viscosity increasing agent, e.g., CMC-Na, may be present in an amount of
from
about 0.1 % to about 2%, e.g., about 0.7% or about 1.75% of the vehicle (by
volume), e.g., in
a concentration of about 1 mg/mL to about 30 mg/mL in the vehicle, e.g., about
7 mg/mL or
about 17.5 mg/mL.
In a further aspect, the present invention provides a kit comprising
microparticles of
the invention and a vehicle of the invention. For example, the kit may
comprise micro-
particles comprising the exact amount of compound of the invention to be
administered, e.g.,
as described below, and about 1 mL to about 5 mL, e.g., about 2 mL of the
vehicle of the
invention.
In one embodiment, the dry microparticles, optionally in admixture with an
anti-
agglomerating agent, may be filled into a container, e.g., a vial or a
syringe, and sterilized
e.g., using gamma-irradiation. Prior to (usually s.c. or i.m.) administration,
the microparticles
may be suspended in the container by adding a suitable vehicle, e.g., the
vehicle described
above. For example, the microparticles, optionally in admixture with an anti-
agglomerating
agent, a viscosity increasing agent and/or a tonicity agent, and the vehicle
for suspension
may be housed separately in a double chamber syringe. A mixture of the
microparticles with
an anti-agglomerating agent and/or a viscosity increasing agent and/or a
tonicity agent, also
forms part of the invention.
In another embodiment, under sterile conditions dry sterilized microparticles,
optio-
nally in admixture with an anti-agglomerating agent, may be suspended in a
suitable vehicle,
e.g., the vehicle described above, and filled into a container, e.g., a vial
or a syringe. The
solvent of the vehicle, e.g., the water, may then be removed, e.g., by freeze-
drying or evapo-
ration under vacuum, leading to a mixture of the microparticles and the solid
components of
the vehicle in the container. Prior to administration, the microparticles and
solid components
of the vehicle may be suspended in the container by adding a suitable vehicle,
e.g., water,
e.g., water for infusion, or preferably a low molarity phosphate buffer
solution. For example,
the mixture of the microparticles, optionally the anti-agglomerating agent,
and solid com-
ponents of the vehicle and the vehicle for suspension, e.g., water, may be
housed separately
in a double chamber syringe.
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III. Implants
It has been found that administration of implants comprising a poorly soluble
salt of a
bisphosphonate of the formula I embedded in a biocompatible pharmacologically
acceptable
polymer gives release of all or of substantially all of the active agent over
an extended period
of time, e.g., one week up to 18 months, especially for about 3 weeks to about
12 months,
e.g. 3 months to about 12 months. The term "depot formulation" in the present
disclosure
therefore also refers to such implants.
The present invention in another aspect provides a process for the preparation
of the
implants of the invention comprising:
(i) preparation of a powder mixture of the poorly water soluble DS and the
biodegradable polymer by cryo-milling with liquid nitrogen of both components
together and/or using a organic solvent for a granulation step and removing
this
solvent again by a drying process;
(ii) filling a RAM extruder with the powder mixture (alternatively, a screw or
a double-
screw extruder is used);
(iii) heating the extruder walls to temperatures in the range of 50-120 C, in
case of
using poly(lactide-co-glycolide) as polymer matrix preferably 60-90 C;
(iv) pushing the molten powder mixture through a pin hole of 1-4 mm diameter
at
small speed, preferably through a 1.5 mm pin hole with a speed of 5 mm/min.;
and
(v) cutting the resulting sticks into shorter length depending on the
anticipated dose,
e.g., 20 mm.
For application the implants are placed in an applicator or trochar, sealed in
aluminum foil and sterilized by using gamma-irradiation with a minimum dose of
25 kGy.
These applicators are commercially available, e.g., by Rexam Pharma,
Suddeutsche
Feinmechanik GmbH (SFM) or Becton Dickerson.
IV. Biocompatable Polymers
The polymer matrix of the depot formulations may be a synthetic or natural
polymer.
The polymer may be either a biodegradable or non-biodegradable or a
combination of
biodegradable and non-biodegradable polymers, preferably biodegradable.
By "polymer" is meant an homopolymer or a copolymer.
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Suitable polymers include:
(a) linear or branched polyesters which are linear chains radiating from a
polyol
moiety, e.g., glucose, e.g., a polyester, such as D-, L- or racemic polylactic
acid,
polyglycolic acid, polyhydroxybutyric acid, polycaprolactone, polyalkylene
oxalate,
polyalkylene glycol esters of an acid of the Kreb's cycle, e.g., citric acid
cycle, and the
like or a combination thereof,
(b) polymers or copolymers of organic ethers, anhydrides, amides and
orthoesters,
including such copolymers with other monomers, e.g., a polyanhydride, such as
a
copolymer of 1,3-bis-(p-carboxyphenoxy)-propane and a diacid, e.g., sebacic
acid, or
a copolymer of erucic acid dimer with sebacic acid; a polyorthoester resulting
from
reaction of an ortho-ester with a triol, e.g., 1,2,6-hexanetriol, or of a
diketene acetal,
e.g., 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5,5]un-decane, with a diol,
e.g.,
1,6-dihexanediol, triethyleneglycol or 1,10-decanediol; or a polyester amide
obtained
with an amide-diol monomer, e.g., 1,2-di-(hydroxyacetamido)-ethane or 1,10-di-
(hydroxyacetamido)decane; or
(c) polyvinylalcohol.
The polymers may be cross-linked or non-cross-linked, usually not more than
5%,
typically less than 1%.
Preferred are polylactide-co-glycolide polymers (also named PLGA).
Table II lists examples of the polymers of the invention:
Table II
Product Name Polymer
D,L-POLYMI/D-GLUCOSE Star-branched
Poly(D,L-lactide-co-glycolide)
50:50 / D-Glucose
Resomer R 202 H Linear
Poly(D,L-lactide)
free carboxylic acid end group
Resomer R 202 Linear
Pol D,L-lactide
Resomer R 203 Linear
Pol D,L-lactide
Resomer RG 752 Linear
Poly(D,L-lactide-co-glycolide)
75:25
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Product Name Polymer
Resomer RG 753 S Linear
Poly(D, L-lactide-co-glycolide)
75:25
Lactel 100D020A Linear
Poly(D,L-lactide)
free carboxylic acid end group
Lactel 100D040A Linear
Poly(D, L-lactide)
free carboxylic acid end group
Lactel 100D040 Linear
Pol D, L-lactide
Lactel 100D065 Linear
Pol D,L-lactide
Lactel 85DG065 Linear
Poly(D,L-lactide-co-glycolide)
85:15
Lactel 75DG065 Linear
Poly(D, L-lactide-co-glycolide)
75:25
Lactel 65DG065 Linear
Poly(D, L-lactide-co-glycolide)
65:35
Lactel 5ODG065 Linear
Poly(D, L-lactide-co-glycolide)
50:50
Lactel 50DG085 Linear
Poly(D,L-lactide-co-glycolide)
50:50
Lactel 50DG105 Linear
Poly(D, L-lactide-co-glycolide)
50:50
Medisorb 100 DL HIGH IV Linear
Pol D,L-lactide
Medisorb 100 DL LOW IV Linear
Pol D,L-lactide
Medisorb 8515 DL HIGH IV Linear
Poly(D,L-lactide-co-glycolide)
85:15
Medisorb 8515 DL LOW IV Linear
Poly(D, L-lactide-co-glycolide)
85:15
Medisorb 7525 DL HIGH IV Linear
Poly(D, L-lactide-co-glycolide)
75:25
Medisorb 7525 DL LOW IV Linear
Poly(D,L-lactide-co-glycolide)
75:25
Medisorb 6535 DL HIGH IV Linear
Poly(D, L-lactide-co-glycolide)
65:35
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Product Name Polymer
Medisorb 6535 DL LOW IV Linear
Poly(D, L-Iactide-co-glycolide)
65:35
Medisorb 5050 DL HIGH IV Linear
Poly(D, L-lactide-co-glycolide)
50:50
Medisorb 5050 DL LOW IV Linear
Poly(D, L-lactide-co-glycolide)
50:50
The preferred polymers of this invention are linear polyesters and branched
chain
polyesters. The linear polyesters may be prepared from alpha-hydroxy
carboxylic acids,
e.g., lactic acid and/or glycolic acid, by condensation of the lactone dimers.
The preferred
polyester chains in the linear or branched (star) polymers are copolymers of
the alpha-
carboxylic acid moieties, lactic acid and glycolic acid, or of the lactone
dimmers, also
referred to herein as PLGA. The molar ratio of lactide: glycolide of
polylactide-co-glycolides
in the linear or branched polyesters is preferably from about 100:0 to 40:60,
more preferred
from. 95:5 to 50:50, most preferred from 95:5 to 55:45.
Linear polyesters, e.g., linear polylactide-co-glycolides, preferably used
according to
the invention have a weight average molecular weight (Mw) between about 10,000
Da and
about 500,000 Da, e.g., about 50,000 Da. Such polymers have a polydispersity
MW/Mn, e.g.,
between 1.2 and 2. Suitable examples include, e.g., poly(D,L-lactide-co-
glycolide), linear
poly (D,L-lactide) and liner-poly (D,L-lactide) free carboxylic acid end
group, e.g., having a
general formula -[(C6H8O4)X(C4H4O4)y]n- (each of x, y and n having a value so
that the total
sum gives the above indicated Mws), e.g., those commercially-available, e.g.,
Resomers
from Boehringer Ingelheim, Lactel from Durect, Purasorb from Purac and
Medisorb from
Lakeshore.
Branched polyesters, e.g., branched polylactide-co-glycolides, also used
according to
the invention may be prepared using polyhydroxy compounds, e.g., polyol, e.g.,
glucose or
mannitol as the initiator. These esters of a polyol are known and described,
e.g., in
GB 2,145,422 B, the contents of which are incorporated herein by reference.
The polyol
contains at least 3 hydroxy groups and has a molecular weight of up to 20,000
Da, with at
least 1, preferably at least 2, e.g., as a mean 3 of the hydroxy groups of the
polyol being in
the form of ester groups, which contain poly-lactide or co-poly-lactide
chains. Typically 0.2%
glucose is used to initiate polymerization. The branched polyesters (Glu-PLG)
have a
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central glucose moiety having rays of linear polylactide chains, e.g., they
have a star shaped
structure.
The branched polyesters having a central glucose moiety having rays of linear
polylactide-co-glycolide chains (Glu-PLG) may be prepared by reacting a polyol
with a lactide
and preferably also a glycolide at an elevated temperature in the presence of
a catalyst,
which makes a ring opening polymerization feasible.
The branched polyesters having a central glucose moiety having rays of linear
polylactide-co-glycolide chains (Glu-PLG) preferably have an weight average
molecular
weight M, in the range of from about 10,000-200,000, preferably 25,000-
100,000, especially
35,000-60,000, e.g., about 50,000 Da, and a polydispersity, e.g., of from 1.7-
3.0, e.g.,
2.0-2.5. The intrinsic viscosities of star polymers of M,, 35,000 or M,,
60,000 are 0.36 dL/g or
0.51 dL/g, respectively, in chloroform. A star polymer having a M,N 52,000 has
a viscosity of
0.475 dl/g in chloroform.
The desired rate of degradation of polymers and the desired release profile
for
compounds of the invention may be varied depending on the kind of monomer,
whether a
homo- or a copolymer or whether a mixture of polymers is employed.
V. Method of Treatment
The uses and methods of the present invention represent an improvement to
existing
therapy of various diseases, including diseases or disorders where abnormal
(especially
abnormally increased) bone turnover is found, also malignant diseases in which
bisphos-
phonates are used to prevent or inhibit development of bone metastases or
excessive bone
resorption, and also especially for the therapy of inflammatory diseases such
as rheumatoid
arthritis and osteoarthritis. Use of bisphosphonates to embolise newly-formed
blood vessels
has been found to lead to suppression of tumors, e.g., solid tumors, and
metastastes, e.g.,
bone metastases and even reduction in size of tumors, e.g., solid tumors, and
metastases,
e.g., bone metastases, after appropriate periods of treatment. It has been
observed using
angiography that newly-formed blood vessels disappear after bisphosphonate
treatment, but
that normal blood vessels remain intact. Further it has been observed that the
embolised
blood vessels are not restored following cessation of the bisphosphonate
treatment. Also it
has been observed that bone metastasis, rheumatoid arthritis and
osteoarthritis patients
experience decreased pain following bisphosphonate treatment.
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Conditions of abnormal, e.g. abnormally increased, bone turnover which may be
treated in accordance with the present invention include: treatment of (e.g.
bone) cancer
related abnormal bone turnover, treatment of postmenopausal osteoporosis,
e.g., to reduce
the risk of osteoporotic fractures; prevention of postmenopausal osteoporosis,
e.g.,
prevention of postmenopausal bone loss; treatment or prevention of male
osteoporosis;
treatment or prevention of corticosteroid-induced osteoporosis and other forms
of bone loss
secondary to or due to medication, e.g., diphenylhydantoin, thyroid hormone
replacement
therapy; treatment or prevention of bone loss associated with immobilisation
and space
flight; treatment or prevention of bone loss associated with rheumatoid
arthritis,
osteogenesis imperfecta, hyperthyroidism, anorexia nervosa, organ
transplantation, joint
prosthesis loosening, and other medical conditions. For example, such other
medical
conditions may include treatment or prevention of periarticular bone erosions
in rheumatoid
arthritis; treatment of osteoarthritis, e.g., prevention/treatment of
subchondral osteosclerosis,
subchondral bone cysts, osteophyte formation, and of osteoarthritic pain,
e.g., by reduction
in intra-osseous pressure; treatment or prevention of hypercalcemia resulting
from excessive
bone resorption secondary to hyperparathyroidism, thyrotoxicosis, sarcoidosis,
or hyper-
vitaminosis D, dental resorptive lesions, pain associated with any of the
above conditions,
particularly, osteopenia, Paget's disease, osteoporosis, rheumatoid arthritis,
osteoarthritis.
Especially useful (for human and veterinary use) is the treatment of one or
more diseases
(this term including conditions or disorders), involving abnormal bone
turnover associated
with diseases of bones and joints, for example
- benign conditions such as osteoporosis, osteopenia, osteomyelitis,
osteoarthritis,
rheumatoid arthritis, bone marrow edema, bone pain, reflex sympathetic
dystrophy,
ankylosing spondylitis (aka Morbus Bechterev), Paget's disease of bone or
periodontal disease,
- malignant conditions such as hypercalcemia of malignancy, bone metastases
associated with solid tumors and hematologic malignancies,
- orthopedic conditions such as prosthesis loosening, prosthesis migration,
implant
fixation, implant coating, fracture healing, distraction osteogenesis, spinal
fusion,
avascular osteonecrosis, bone grafting, bone substitutes,
or any combination of tow or more such conditions.
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Appropriate dosage of the depot formulations of the invention will of course
vary,
e.g., depending on the condition to be treated (e.g., the disease type or the
nature of
resistance), the drug used, the effect desired and the mode of administration.
Specifically, with a depot formulation according to the invention satisfactory
results
are obtained on administration, e.g., parenteral administration, at dosages on
the order of
from about 0.2 mg to about 100 mg, e.g., 0.2 mg to about 35 mg, preferably
from about 3
mg to about 100 mg of the compound of the formula I (calculated based on its
free form) of
the invention per injection per month or about 0.03 mg to about 1.2 mg, e.g.,
0.03-0.3 mg
per kg animal body weight per month. Suitable monthly dosages for patients are
thus in the
order of about 0.3 mg to about 100 mg of a compound of the formula I
(calculated based on
its free form, also here it is used in the form or the salt and/or crystal).
The pharmaceutical compositions in more general form which contain a compound
of
formula I as crystalline form of the free (e.g. zwitterionic) form or a poorly
soluble salt of a
compound of the formula I or especially a crystalline form of such a salt
(ncluding a solvate,
e.g. hydrate, especially a dihydrate, of such salt) as decribed hereinabove
and -below are
those for enteral such as oral, or rectal and parenteral, administration to
warm-blooded
animals, the pharmacological active ingredient being present alone or together
with a
pharmaceutically suitable carrier.
These further novel pharmaceutical compositions comprise e.g. from about
0.0001 to 80%,
preferably from about 0.001 to 10%, of the active ingredient. Pharmaceutical
compositions
for enteral or parenteral administration are e.g. those in dosage unit forms
such as dragees,
tablets, capsules or suppositories, as well as ampoules, vials, pre-filled
syringes. These
pharmaceutical compositions are prepared in a manner known per se, for example
by con-
ventional mixing, granulating, confectioning, dissolving or lyophilising
methods. For example,
pharmaceutical compositions for oral administration can be obtained by
combining the active
ingredient with solid carriers, optionally granulating a resulting mixture and
processing the
mixture or granulate, if desired or necessary after the addition of suitable
excipients, to
tablets or dragee cores.
Suitable carriers are in particular fillers such as sugar, for example
lactose, saccharose,
mannitol or sorbitol, cellulose preparations and/or calcium phosphates, e.g.
tricalcium phos-
phate or calcium biphosphate, and also binders such as starch pastes, e.g.
maize, corn, rice
or potato starch, gelatin, tragacanth, methyl cellulose and/or
polyvinylpyrrolidone, and/or, if
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desired, disintegrators, such as the abovementioned starches, also
carboxymethyl starch,
crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as
sodium alginate.
Excipients are in particular glidants and lubricants, for example silica,
talcum, stearic acid or
salts thereof such as magnesium stearate or calcium stearate, and/or
polyethylene glycol.
Dragee cores are provided with suitable coatings which can be resistant to
gastric juices,
using inter alia concentrated sugar solutions which may contain gum arabic,
talcum, polyvi-
nylpyrrolidone, polyethylene glycol and/or titanium dioxide, shellac solutions
in suitable orga-
nic solvents or mixtures of solvents or, for the preparation of coatings which
are resistant to
gastric juices, solutions of suitable cellulose preparations such as acetyl
cellulose phthalate
or hydroxypropyl methyl cellulose phthalate. Dyes or pigments can be added to
the tablets or
dragee coatings, for example to identify or indicate different doses of active
ingredient.
Further pharmaceutical compositions for oral administration are dry-filled
capsules made of
gelatin or hypromellose and also soft sealed capsules consisting of gelatin
and a plasticiser
such as glycerol or sorbitol. The dry-filled capsules can contain the active
ingredient in the
form of granules, for example in admixture with fillers such as lactose,
binders such as star-
ches, and/or glidants such as talcum or magnesium stearate, and optionally
stabilisers. In
soft capsules, the active ingredient is preferably dissolved or suspended in a
suitable liquid,
such as a fatty oil, paraffin oil or a liquid polyethylene glycol, to which a
stabiliser can also be
added.
Suitable pharmaceutical compositions for rectal administration are e.g.
suppositories, which
consist of a combination of the active ingredient with a suppository base.
Examples of suit-
able suppository bases are natural or synthetic triglycerides, paraffin
hydrocarbons, poly-
ethylene glycols and higher alkanols. It is also possible to use gelatin
rectal capsules which
contain a combination of the active ingredient with a base material. Suitable
base materials
are e.g. liquid triglycerides, polyethylene glycols and paraffin hydrocarbons.
Particularly suitable dosage forms for parenteral administration (which is
especially prefer-
red) are aqueous solutions of an active ingredient in water-soluble form, for
example a
water-soluble salt. The solution may be adjusted with inorganic or organic
acids or bases to
a physiologically acceptable pH value of about pH 4-9 or most preferably of
about 5.5 - 7.5.
The solutions further may be made isotonic with inorganic salts like sodium
chloride, or
organic compounds like sugars, sugar alcohols, or amino acids, most preferably
with
mannitol or glycerol. Suitable compositions are also suspensions of the active
ingredient,
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such as corresponding oily injection suspensions, for which there are used
suitable lipophilic
solvents or vehicles such as fatty oils, for example sesame oil, or synthetic
fatty acid esters,
for example ethyl oleate or triglycerides, or aqueous injection suspensions
which contain
substances which increase the viscosity, for example sodium carboxymethyl
cellulose,
sorbitol and/or dextran, and optionally also stabilisers.
The present invention also relates to the forms of the compound of the formula
I (including a
salt, a crystalline form, and/or a depot formulation) preferably for the
treatment of
inflammatory conditions, primarily to diseases associated with impairment of
calcium
metabolism, e.g. rheumatic diseases and, in particular, osteoporosis.
Parenteral Doses below 0.1 pg/kg of body weight affect hard tissue metabolism
only
insignificantly. Long-term toxic side-effects may occur at doses of over 1000
pg/kg of body
weight. The forms of compounds of formula I according to the invention can be
administered
orally, as well as subcutaneously, intramuscularly or intravenously in iso- or
hypertonic
solution. Preferred daily doses are, for oral administration, in the range
from about 1 to 100
mg/kg, for intravenous, subcutaneous and intramuscular administration in the
range from
about 20 to 500 pg/kg.
The dosage of the forms of compounds of formula I (based on weight of the
compound of
formula I as such), however, variable and depends on the respective conditions
such as the
nature and severity of the illness, the duration of treatment and on the
respective compound.
Dosage unit form for parenteral, e.g. intravenous, administration contain e.g.
from 10 to 300
pg/kg of body weight, preferably from 15 to 150 pg/kg body weight; and oral
dosage unit
forms contain e.g. from 0.1 to 5 mg, preferably from 0.15 to 3 mg per kg body
weight. The
preferred single dose for oral administration is from 10 to 200 mg and, for
intravenous
administration, from I to 10 mg. The higher doses for oral administration are
necessary on
account of the limited absorption. In prolonged treatment, the dosage can
normally be
reduced to a lower level after an initially higher dosage in order to maintain
the desired
effect. Parenteral, (e.g. intravenous or subcutaneous) doses may be
administered
intermittently at regular intervals between 1 and 52 times per year. Oral
doses may be
administered regularly on a daily, weekly, monthly or quarterly dosing
regimen. For depot
formulations according to the invention the dosages mentioned above are
preferred.
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The properties of the depot formulations, salts, crystal forms and
pharmaceutical
compositions of the invention may be tested in standard animal tests or
clinical trials, e.g. as
following:
The following publications (each of which is incorporated herein by reference,
especially with
regard to the description of the assays or methods mentioned below therein)
describe
various assays and methods that can be used to confirm the advantageous
biological profile
of the compounds of the formula I:
The effects of a single i.v. administration to mature, ovariectomized (OVX)
rats as a model
for postmenopausal osteoporosis in order to elucidate (1) the temporal changes
in
biochemical markers of bone turnover and femoral bone mineral density (BMD),
(2) to
measure changes of static and dynamic histomorphometric parameters, bone micro-
architecture and mechanical strength, and (3) to assess the preventive effects
of chronic
treatment with a compound of the formula I on these parameters can be
demonstrated as
described in Calcif. Tissue Int. (2003) 72, 519-527. High activity can be
found here.
The effect of a compound of the formula I (in the description of the following
descriptions of
possible biological assays this includes one or both of the salt forms as well
as the crystal
forms described herein) on synovial inflammation, structural joint damage, and
bone
metabolism in rats during the effector phase of collagen-induced arthritis
(CIA) can be
demonstrated as shown in ARTHRITIS & RHEUMATISM (2004), 50(7), 2338-2346.
The effect of a compound of the formula I on bone ingrowth can be examined in
an animal
model in which porous tantalum implants are placed bilaterally within the
ulnae of dogs as
described in J. Bone Joint Surg. (2005), 87-B, 416-420.
Inhibition of skeletal tumor growth in a mouse model can be demonstrated in
accordance
with the method described in J. NatI. Cancer. Inst. (2007), 99, 322 - 30.
The x-ray structure of compounds of the formula I when bound to farnesyl
pyrophosphate
synthase can be obtained by or in analogy to the methods described in Chem.
Med. Chem.
(2006), 1, 267 - 273. Human FPPS, a homodimeric enzyme of 41-kDa subunits,
catalyzes
the two-step synthesis of the C15 metabolite farnesyl pyrophosphate (FPP) from
the C5
isoprenoids dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate.
FPP is
required for the posttranslational prenylation of essential GTPase signalling
proteins such as
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Ras and Rho and is also a precursor for the synthesis of cholesterol,
dolichol, and ubiqui-
none.
For example, in a cell-free in vitro assay the superiority of compounds of the
formula I over
compounds already known can be shown. Briefly, the reaction proceeds in the
presence of
enzyme and an inhibitor of the formula I, and the reaction product (farneysyl
pyrophosphate)
is quantified by LC/MS/MS.
In detail, the inhibitor and enzyme are pre-incubated before adding the
substrates
The assay is a label-free assay for farnesyl pyrophosphate synthase (FPPS)
based on
LC/MS/MS. This method quantifies in-vitro untagged farnesyl pyrophosphate
(FPP) and is
suitable for high throughput screening (HTS) to find inhibitors of FPPS and
for the
determinations of IC50 values of candidate compounds. The analysis time is 2.0
minutes
with a total cycle time of 2.5 minutes. The analysis can be formatted for 384-
well plates
resulting in an analysis time of 16 hours per plate.
Reagents:
Pentanol, methanol, and isopropyl alcohol are HPLC grade and obtained from
Fisher
Scientific. DMIPA is from Sigma-Aldrich. Water is from an in-house Milli-Q
system. The
assay buffer (20 mM HEPES, 5 mM MgC12 and 1 mM CaCl2) is prepared by dilution
from 1
mM stock solutions obtained from Sigma-Aldrich. Standards of geranyl
pyrophosphate
(GPP), isoprenyl pyrophosphate (FPP), and farnesyl S-thiolopyrophosphate
(FSPP) are from
Echelon Biosciences (Salt Lake City, UT). Human farnesyl pyrophosphate
synthase (FPPS,
Swissprot ID: P14324) (13.8 mg/mL) is prepared as described by Rondeau et al
(ChemMedChem 2006, 1, 267-273.
Assay:
LC/MS/MS analyses are performed on a Micromass Quattro Micro tandem quadrupole
mass
analyser (Waters Corp., Milford, MA, USA) interfaced to an Agilent 1100 binary
LC pump
Agilent Technologies, Inc., Santa Clara, CA, USA). Injection is performed with
a CTC
Analytics autosampler (Leap Technologies Inc., Carrboro, NC, USA) using an
injection loop
size of 2.5 pL. Chromatography is performed on a Waters 2.1 x 20 mm Xterra MS
C18 5 pm
guard column (P/N186000652) (Waters Corp., Milford, MA, USA) contained in a
guard
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column holder (P/N 186000262) using 0.1% DMIPA/methanol as solvent A and 0.1%
DMIPA/water as solvent B (DMIPA is dimethylisopropylamine). The gradient is 5%
A from
0.00 to 0.30 min., 50% A at 0.31 min., 80% A at 1.00 min., and 5% A from 1.01
to 2.00 min.
The flow rate is 0.3 mL/min, and the flow is diverted to waste from 0.00 to
0.50 min and
again from 1.20 to 2.00 min.
The Multiple Reaction Monitoring (MRM) transitions monitored are 381->79- for
FPP and
397->159- for FSPP at a collision energy of 22 eV and a collision cell
pressure of 2.1 x 10-3
mbar of Ar. The dwell time per transition is 400 msec with a span of 0.4 Da.
The inter-
channel delay and interscan delay are both 0.02 sec. Other mass spectrometric
operating
parameters are: capillary, 2.0 kV; cone, 35 V; extractor, 2.0 V, source temp.,
100 C;
desolvation gas temp., 250 C; desolvation gas flow, 650 L/hr; cone gas flow,
25 L/hr;
multiplier, 650 V.
The total cycle time per sample is 2.5 minutes. Since the analysis is
formatted for 384-well
plates, a plate is analyzed in 16 hours. The chromatograms are processed using
Quanlynx
software, which divides the area of individual FPP peaks by the area of the
FSPP peaks
(internal standard). The resulting values are reported as the relative
response for the
corresponding sample well.
FPPS Assay Procedure
Into each well of a 384-well plate, 5 pL of compound in 20% DMSO/water is
placed. 10 pL
of FPPS (diluted 1 to 80000 with assay buffer) is added to each well and
allowed to pre-
incubate with the compound for 5 minutes. At that time, 25 pL of GPP/IPP (5 pM
each in
assay buffer) is then added to start the reaction. After 30 minutes the
reaction is stopped by
addition of 10 pL of 2 pM FSPP in 2% DMIPA/IPA. The reaction mixture is then
extracted
with 50 pL of n-pentanol using vortex mixing. After phase separation, 25 pL of
the upper (n-
pentanol) layer is transferred to a new 384-well plate and the pentanol is
evaporated using a
vacuum centrifuge. The dried residue is reconstituted in 50 pL of 0.1%
DMIPA/water for
analysis by the LC/MS/MS method.
FSPP is used as the internal standard for the mass spectra. A phosphate moiety
generates
an (M-H)- ion as the base peak in the spectra.
The compounds of the invention preferably, in this test system, have an IC50
in the range
from 0.8 to 10 nM, the preferred ones preferably from 0.9 to 3.3 nM, (e.g. in
the case of
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experiments with [2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1-phosphono-ethyl]-
phosphonic acid in
the range from 2.4 to 3.1 nM). Especially, they, e.g. [2-(5-ethyl-imidazol-1-
yl)-1-hydroxy-1-
phosphono-ethyl]-phosphonic acid, show a surprising superiority over compounds
in the prior
art.The utility of the assay for IC50 determinations is validated using
zoledronic acid, a known
bisphosphonate inhibitor of FPPS.
The depot formulation, the salts and the crystal forms, as well as the
compositions of the
invention, are well-tolerated.
The invention also relates to the embodiments given in the claims, especially
the dependent
claims, so that said claims are incorporated here by reference, as well as
especially to the
embodiments of the invention provided in the following Examples.
Also the Abstract is incorporated here by reference, also disclosing
embodiments of the
invention.
General preparation of compounds of the formula I:
A compound of the formula I can be prepared according to methods that, for
different
compounds, are known in the art. For example, based at least on the novel
products
obtained and/or the novel educts employed, a novel process is preferred
comprising reacting
a carboxylic acid compound of the formula II,
Ri
OH
R2 / N '_~r
-N J O
(II)
wherein R, and R2 are as defined for a compound of the formula I, with
phosphorous
oxyhalogenide to give a compound of the formula I, or a salt thereof,
and, if desired, converting an obtainable free compound of the formula I into
its salt, con-
verting an obtainable salt of a compound of the formula I into the free
compound and/or
converting an obtainable salt of a compound of the formula I into a different
salt thereof.
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As phosphorous oxyhalogenide, phosphorous oxychloride (POC13) is especially
preferred.
The reaction preferably takes place in a customary solvent or solvent mixture,
e.g. in an aro-
matic hydrocarbon, such as toluene, at preferably elevated temperatures, e.g.
in the range
from 50 C to the reflux temperature of the reaction mixture, e.g. from
(about) 80 to (about)
120 C.
The starting materials of the formula II can, for example preferably, be
obtained by
saponifying a compound of the formula III,
Ri
OR
R2 N
N J O
(III)
wherein R, and R2 are as defined for a compound of the formula I and R is
unsubstituted or
substituted alkyl, especially lower alkyl or phenyl-lower alkyl, in the
presence of an
appropriate acid, e.g. a hydrohalic acid, such as hydrochloric acid,
preferably in the presence
of an aqueous solvent, such as water, at preferably elevated temperatures,
e.g. in the range
from (about) 50 to (about) 100 C, e.g. from 80 to 100 C, to give the
compound of the
formula 11, or a salt thereof.
A compound of the formula III can, for example preferably, be obtained by
reacting an
imidazole compound of the formula IV,
Ri
R2-_~~ NH
N J (IV)
wherein R, and R2 are as defined for a compound of the formula I, with an
ester of the
formula V,
X~ OR
(V)
0
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wherein R is as defined for a compound of the formula III and X is halogen,
especially fluoro,
chloro, iodo or especially bromo, lower-alkanesulfonyloxy or
toluenesulfonyloxy, preferably in
the presence of a strong base, such as an alkaline metal alcoholate,
especially potassium
tert-butylate, in an appropriate solvent or solvent mixture, e.g. a cyclic
ether, such as
tetrahydrofurane, preferably at temperatures in the range from (about) -10 to
(about) 80 C,
e.g. from 20 to 30 C. Where required, resulting mixtures of compounds of the
formula III
(wherein in one compound R, is C2-C5-alkyl and R2 is hydrogen, in the other R2
is C2-C5-alkyl
and R, is hydrogen) can be separated e.g. by chromatographic methods,
differential
crystallisation or the like.
Starting materials of the formulae IV and V, as well as any other starting
materials employed
not described so far, can be obtained by methods that are known in the art or
in analogy
thereto, are commercially available and/or can be made in analogy to methods
described
herein.
The following Examples serve to illustrate the invention without limiting its
scope.
The following compounds of the formula I are obtained as described in the
following
Reference Examples:
If not mentioned otherwise, temperatures are given in degree Celsius ( C).
Where no
temperature is mentioned, the reaction or other method step takes place at
room
temperature.
Abbreviations:
Ac. acetyl
aq. Aqueous
DMSO dimethyl sulfoxide
Et ethyl
h hour(s)
HPLC high performance liquid chromatography
KOtBu potassium tert-butylate
Me methyl
ml milliliter(s)
NMR Nuclear Magnetic Resonance
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rt room temperature
THE tetrahydrofurane
4- and 5-Ethylimidazole and all other imidazole derivatives are prepared
according to D.
Horne et al., Heterocycles, 1994, Vol. 39, No. 1, p.139-153.
Reference-Example 1: f2-(4-Ethyl-imidazol-1-yi)-1-hydroxy-1-phosphono-ethyll-
phosphonic
acid (also named Compound A or Cpd. A hereinafter)
650 mg (3.38 mmol) (4-ethyl-imidazol-1-yl)-acetic acid are dissolved in 15 ml
toluene at it
under nitrogen. 852 mg (3 mmol) H3PO3 are added and the mixture is heated to
80 C. 0.936
ml (3 mmol) POC13 are added drop wise. The resulting mixture is heated to 120
C and stirred
overnight. The solvent is decanted off, 15 ml 6N HCI is added and the mixture
is heated for
three hours at reflux.
The resulting pale yellow solution is concentrated in vacuo. After dilution
with acetone (25
ml) the mixture is stirred vigorously with acetone (5 x 25 ml) until a grey
solid is formed. The
grey solid is dried in high vacuo and crystallized from EtOH/water to give the
title compound.
HPLC-MS: t = 0.31 min, (M-H)- = 299; 'H-NMR (D20/NaOD): 6 = 1.07 (t, 3H), 2.53
(q, 2H),
4.45 (t, 2H), 7.08 (s, 1 H), 8.40 (s, 1 H), 31P-NMR (D2O/NaOD): 6 = 15.04 ppm
Synthesis overview:
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N`
N
H
KOtBu, THF, 25 C,
3h B r
O
N/N N/N
O O
0 4N HCI, 1000C,
HO,,\\ OH 2h
HOOP /O H3PO3, POC13 r>~ P/ toluene, 80 C-1200C
\OH 16h
N OH N
N
N /~--O H
O
HPLC-MS conditions:
Column: XTerra (Waters Corp., Milford, MA, USA) 3x30 mm, 2.5pm, C18
Solvent A: water, 5% acetonitrile, 1% HCOOH
Solvent B: acetonitrile, 1% HCOOH
Gradient: min % B
0,0 01
0,5 01
2,5 30
3,5 95
4,5 95
4,9 01
The starting materials are prepared as follows:
Step 1: (4-Ethyl-imidazol-1-yl)-acetic acid ethyl ester and (5-ethyl-imidazol-
1-yl)-acetic
acid ethyl ester
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5.02 g (50 mmol) of 4-ethylimidazole are dissolved in 100 ml THE at rt under
nitrogen. 5.9 g
(52 mmol) KOtBu is added and the reaction is stirred for 2h at it. 6.3 ml (55
mmol) ethyl
bromoacetate is added drop wise over a period of 30 min and the resulting
mixture is stirred
at it for 2.5 h. 20 ml H2O and 130 ml AcOEt are added, the organic layer is
separated and
the aq. layer is washed again 2 x with 100 ml AcOEt. The combined organic
layer is washed
with brine, dried over MgSO4 and concentrated in vacuo. The reaction is
purified by Flash-
chromatography (silica gel, MeOH/methylen chloride) to give (4-ethyl-imidazol-
1-yl)-acetic
acid ethyl ester and (5-ethyl-imidazol-1-yl)-acetic acid ethyl ester,
respectively.
(4-Ethyl-imidazol-1-yl)-acetic acid ethyl ester: HPLC-MS: t = 0.60 min; 100
area%, MH+=
183; 1H-NMR (d6-DMSO) b = 1.09 (t, 3H), 1.18 (t, 3H), 2.43 (q, 2H), 4.13 (q,
2H), 4.83 (s, 2
H), 6.78 (s, 1 H), 7.43 (s, 1 H)
(5-Ethyl-imidazol-1-yl)-acetic acid ethyl ester: HPLC-MS : t = 0.72 min, 100
area%,
MH+=183; 1H-NMR (d6-DMSO): b = 1.12 (t, 3H), 1.18 (t, 3H), 2.40 (q, 2H), 4.14
(q, 2H), 4.85
(s, 2H), 6.61 (s, 1 H), 7.48 (s, 1 H)
Step 2: (4-Ethyl-imidazol-1-yl)-acetic acid
1.7 g (9.5 mmol) of (4-ethyl-imidazol-1-yl)-acetic acid ethyl ester are
dissolved in 47 ml (190
mmol) 4N HCI and the mixture is heated to reflux. After 2h the mixture is
cooled to it and the
solvent is removed in vacuo. The resulting product is used without further
purification. MS:
MH+= 155, 1H-NMR (DMSO): b = 1.18 (t, 3H), 2.65 (q, 2H), 5.07 (s, 2H), 7.43
(d, 1H). 9.0 (d,
1 H)
Reference-Example 2: [2-(5-Ethyl-imidazol-l-yl)-1-hydroxy-l-phosphono-ethyll-
phosphonic
acid
[2-(5-Ethyl-imidazol-1-yl)-l-hydroxy-1-phosphono-ethyl]-phosphonic acid is
synthesized
according to the synthesis outlined above from the corresponding (5-ethyl-
imidazol-1-yl)-
acetic acid ethyl ester which is the second product of step 1 in Example 1.
OHOH
O '
p
OH
~,~* N
0=P
N /
HO OH
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HPLC-MS: t = 0.32 min, (M-H)- = 299; 1H-NMR (D20/NaOD): 8 = 1 .10 (t, 3H),
2.63 (q, 2H), 4.43 (t, 2H), 6.95 (s, 1 H), 8.54 (s, 1 H), 31P-NMR (D20/NaOD):
8
= 14.96 ppm
In analogy to the above described procedures the following compounds are
prepared:
Reference-Example 3: [2-(4-Propyl-imidazol-1-yl)-1-hvdroxv-1-phosphono-ethyll-
phosphonic
acid
HO OH
OH
O=P
N P\ OH
O
OH
N
HPLC-MS: t = 0.44 min, (M-H)- = 313.1; 1H-NMR (D20/NaOD): b = 0.78 (t, 3H),
1.52 (m,
2H), 2.52 (t, 2H), 4.50 (t, 2H)7.13 (s, 1 H), 8.45 (s, 1 H); 31P-NMR
(D20/NaOD) 6 = 15.25 ppm
Reference-Example 4: [2-(5-Propyl-imidazol-1-yl)-1-hvdroxv-1-phosphono-ethyll-
phosphonic
acid
HO
HO ",\ OH
// P
O PSOH
N 0 OH
N
HPLC-MS: t = 0.46 min, (M-H)- = 313.1; 1H-NMR (D20/NaOD): 6 = 0.81 (t, 3H),
1.51 (m,
2H), 2.60 (t, 2H), 4.44 (t, 2H), 6.96 (s, 1H), 8.54 (s, 1H); 31P-NMR
(D20/NaOD) 6 = 15.06
ppm
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Reference-Example 5: f2-(4-Butyl-imidazol-l -yl)-1-hydroxv-l-phosphono-ethyll-
phosphonic
acid
HO OH
OH
0=P
N - P\ OH
I O
OH
N
HPLC-MS: t = 0.56 min, (M-H)- = 327.2; 'H-NMR (D20/NaOD): 6 0.73 (t, 3H), 1.17
(m, 2H),
1.46 (m, 2H), 2.51 (t, 2H), 4.44 (t, 2H ) 7.09 (s, 1 H), 8.40 (s, 1 H) ; 31P-
NMR (D20/NaOD): 6
= 14.98 ppm
Reference-Example 6: f2-(5-Butyl-imidazol-1-yl)-1-hydroxv-1-phosphono-ethyll-
phosphonic
acid
HO
HO I\ OH
// P
O P OH
0 \OH
N
N
HPLC-MS: t = 0.44 min, (M-H)- = 327.2; 1H-NMR (D20/NaOD): 6 = 0.79 (t, 3H),
1.27 (m,
2H), 1.51 (m, 2H), 2.67 (t, 2H), 4.49 (t, 2H), 6.99 (s, 1H), 8.58 (s, 1H) ;
31P-NMR
(D20/NaOD): 6 = 15.16 ppm
Reference-Example 7: f 1-Hydroxy-2-(4-isopropyl-imidazol-1-yl)-1-phosphono-
ethyll-
phosphonic acid
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HO OH
OH
0=P
N - P\ OH
0 OH
f N
HPLC-MS: t = 0.42 min, (M-H)- = 313; 'H-NMR (d6-DMSO): 6 = 1.13, 1.15 (d, 6H),
2.86-
2.95 (m, 1 H), 4.49 (t, 2H), 7.12 (s, 1 H), 8.46 (s, 1 H) ; 31 P-NMR (d6-
DMSO): 6 = 15.35 ppm
Reference-Example 8: [[1-Hydroxy-2-(5-isopropyl-imidazol-l-yl)-1-phosphono-
ethyll-
phosphonic acid
HO
HO '\,\ OH
// P
O P -OH
N 0 OH
N
HPLC-MS: t = 0.40 min, (M-H)- = 313; 'H-NMR (d6-DMSO): 6 = 1.10, 1.12 (d, 6H),
3.12-
3.19 (m, 1 H), 4.52 (t, 2H), 7.01 (s, 1 H), 8.56 (s, 1 H) ; 31 P-NMR (d6-
DMSO): 6 = 15.24 ppm
Reference-Example 9: f(2-f4-(1-Ethyl-propyl)-imidazol-l-yil-l-hydroxy-l-
phosphono-ethyl}-
phosphonic acid
HO OH
OH
0=P
N O P\ OH
1 /~ 0 H
N
HPLC-MS: t = 0.55 min, (M-H)- = 341; 1H-NMR (d6-DMSO): d = 0.80 (m, 6H), 1.50-
1.75 (m,
4H), 2.49-2.60 (m, 1 H), 4.52 (bs, 2H), 7.40 (s, 1 H), 8.90 (s, 1 H).
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Reference-Example 10: {2-f5-(1-Ethyl-propyl)-imidazol-l-vll-1-hydroxy-l-
phosphono-ethyl}-
phosphonic acid
HO
HO 1-1\ OH
/P
0 (OH
3---c O OH
N
'H-NMR (d6-DMSO): d = 0.77 (m, 6H), 1.40-1.60 (m, 4H), 2.97 (t, 1H), (4.44 (t,
2H), 6.60 (s,
1 H), 7.92 (s, 1 H).
Example 1: Manufacturing process for Ca-[2-(5-Ethyl-imidazol-1-yl)-1-hydroxy-1-
phosphono-ethyll-phosphonic acid salt (1 : 2)
3.5 g (11.67 mmol) of [2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1-phosphono-ethyl]-
phosphonic
acid are dissolved in 540 ml of deionized water at 90 C. To this solution,
within 1 minute a
90 C hot solution of 667 mg (5.83 mmol) calcium chloride in 10 ml of water is
added. The
reaction mixture is cooled to 20 C over 14 h and the suspension is filtered.
The solid is
washed with 2 x 50 ml of ice water and dried at 60 C and 5 mbar. The calcium-
[2-(5-ethyl-
imidazol-1-yl)-1 -hydroxy-1 -phosphono-ethyl]-phosphonic acid salt (1:2) is
obtained.
Example 2: Manufacturing process for Zn-[2-(5-Ethyl-imidazol-1-yl)-1-hydroxy-1-
phosphono-ethyll-phosphonic acid salt (1 : 2)
3.5 g (11.67 mmol) of [2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1 -phosphono-ethyl]-
phosphonic
acid are dissolved in 540 ml deionized water at 90 C. To this solution,
within 1 minute a 90
C hot solution of 811 mg (5.83 mmol) zinc chloride is 10 ml of water is added.
The reaction
mixture is cooled to 20 C and dried at 60 C to yield the zinc-[2-(5-ethyl-
imidazol-1-yl)-1-
hydroxy-1-phosphono-ethyl]-phosphonic acid salt (1:2).
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Example 3: Micronisation of the salts from Examples 1 and 2 and manufacturing
of
microparticles according to the invention with the micronized salts
a) Micronisation
(i) Millin
The dry Calcium-salt of Example 1 and the dry Zinc- salt of Example 2 are
milled on an
ceramic air-jetmill (5 bar milling gas pressure).
(ii) Resulting Particles
Before milling of the Calcium salt, the particles have a size of up to about
150 pm. After the
milling, the particles have a size smaller than 10 pm according to microscopy.
In the case of the Zinc salt, the particles before milling have a size of up
to about 100 pm.
After the milling, the particles display sizes below 5 pm.
b) Manufacturing of microparticles
3.1 g of a PLGA 50:50 with an inherent viscosity of 0.38 dL/g (Lactel ) are
dissolved in 15.5
mL dichloromethane to form a clear 20% (mN) PLGA solution. 0.9 g Ca-[2-(5-
ethyl-imidazol-
1-yl)-1-hydroxy-1-phosphono-ethyl]-phosphonic acid (1:2) salt from Example 1
(assay of free
acid 89.0%) are dispersed into the PLGA solution using a high-shear force
mixer (Ultra
Turrax, S25N-10G) at 20'000 rpm for 4 min under cooling in an ice-water bath.
The resulting
suspension is referred to as the organic phase.
25 g of a polyvinyl alcohol 18-88 (PVA hereinafter), 11.3 g of sodium acetate
trihydrate and
25.0 g glacial acetic acid are dissolved in 5 L water. This 0.5% PVA - 100 mM
Acetate buffer
solution with a pH of 4 is referred to as aqueous phase.
The organic phase is mixed with the aqueous phase through an in-line high
shear force
device with two inflows and one outflow at a flow rate ratio of 30 : 600
mL/min and at 3800
rpm. The resulting emulsion is collected in a double walled reactor bearing
already a starting
volume of 170 mL aqueous phase under stirring with a propeller stirrer at 400
rpm.
The dichloromethane is removed by evaporation which is facilitated by
continuous stirring of
the batch with 400 rpm, slowly heating up the batch to 50 C within 5 hours,
keeping this
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temperature for further 2 hours. During all this time the gas phase near the
surface of the
emulsion is exchanged using vacuum.
After cooling down to room temperature again the formed microparticles
sediment for 12
hours. The supernatant is removed to large extend. The microparticles are
resuspended
again in the remaining supernatant and isolated by filtration over 5 pm. The
microparticles
are washed 4 times with ca. 50 mL water and dried in vacuum for 3 days.
Finally, the dried
microparticles are desagglomerated by sieving through 140 pm mesh size. 2.32 g
microparticles are obtained as fine white powder. The electron microscopic
image showed
perfectly spherical particles of smooth surface. The microparticle size
distribution was found
by laser light diffraction as follows: x10: 15.6 pm, x50: 35.8 pm, x90: 53.7
pm. A drug
substance assay of 14.7% is found by HPLC which corresponds to an
encapsulation efficacy
of 74%. After 24 h only 1.9 % of the drug substance is released in an in vitro
release test in a
buffer of pH 7.4 at 37 C, which shows that this formulation advantageously
avoids a too
early release..
The following table summarize the key formulation, process and analytical data
of example 1
and for further examples 2 - 4 of [2-(5-ethyl-imidazol-1-yl)-1-hydroxy-1-
phosphono-ethyl]-
phosphonic acid salt (composition see table) microparticles prepared
analogously to
example 1.
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WO 2009/121935 PCT/EP2009/053965
ti N-
C
x N L'^) M
0
co CO M
f4 N CO
(6 s- LU N
Q)
U)
CD
Q) O
ti N N
Q)
N M
E CO CO N
Q) Co
U) CO 0
N
Q)
Q)
L /^
0) U N-
L
r r r
1 1 1 p
O
01 -O Q) 1 U I` 80- LO
00
L O L U (( O (6
O N- N
00 d7 M
M X LU LO
E
O rn 00
N C LO Ln L6
U O X CO Co
0
O CO 0
X '-
cu LO
N N
0) ,-. M M
N N
yd
a 0 4-
0
0)
C O
C) J i) O `0
0 O N U 0 m U') 00 O N
_ L > < o O D) LO O M N-
4-
0
A U O O
CO Q) O U O
U) (n Q) E rn 0 u) ca C) Q 4- Q O co 0O co
O
Q. 1 1 O 1 U
j, L N C
CE -L -a o 0
N N
N 0 C Q O Q co
O
O O
x n a) a CO CO CO
N E
E L.
0 LL F-
'- (0
(D W a) (0 Q m
CO
H
CA 02720418 2010-10-01
WO 2009/121935 PCT/EP2009/053965
r) co
U) U)
c
O N
L1) LC)
r r
f` O
LO
O
Ln
O T7
CO O
IT LO
LC) Ln
ti T-
d- LO
o
o N o 6) o
M O
M
N O
ti
LO LO
N U)
M N-
M M
T7
ti
00
r N
N co
C) N
a) a)
O -o a) U) J O
LO 00 N- N ti
O M O t?j if
O M U) O M N- O M
O o a)
0) (0 M CO M
U) 0 O
O 00 O 00 O
N N
C
N N
U 0
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DS stands for drugs substance, L:G for the molar ratio of lactic acid to
glycolide in the co-
monomer. In examples 1 and 3 Lactel is used as PLGA, in examples 2 and 4
Resomer RG 753 S (see Table II above).
This shows quite clearly that in this case the Ca-salt microparticle
formulation of Example 3A
shows the most advantageous release kinetics with only very low release on day
1 and less
than 60 % release until day 21 of the compound of the formula I.
Using scanning electron microscopic images (the samples are sputtered with
gold-palladium
and investigated by a scanning electron microscopy of the microparticles of
the formulations
of Example 3A, 3B, 3C and 3D), it becomes evident that in the case of the Zn-
salt particles
(Examples 3C and 3D) the drug substance is apparently not encapsulated at all
or only to a
minor degree: Drug substance particles can be observed on the surface of the
particles, and
no drug substance can be seen in the polymer matrix in cross sections. For the
surface
representation, the microparticles are transferred to suitable object holders,
sputtered with
20 nm gold and examines with the scanning electron microscope (Camscan CS
24/EO, Id.
G.16.MIK.S008). For the cross section views, cross sections are prepared by
embedding the
particles in Araldite F (trademark from Ciba Specialty Chemicals, Basle,
Switzerland; epoxy
resin) and cutting semi-thin sections (thickness approximately 1 pm). The
sections are
sputtered with gold and also examined in the SEM.
In contrast, the images of the Ca salt microparticles (Examples 3A, 3B)
demonstrate the
more efficous encapsulation compared to the Zn salt. However, still some drug
substance
particles can be seen at the surface of the PLGA 75:25 formulation (Example
3B) which
explains the high burst effect (high first 24h release). In contrast, the Ca
salt formulation with
PLGA 50:50 shows no drug substance at all at the surface. In the cross-section
of particles
the drug substance particles can be observed.
Example 4: Tolerability Study of Calcium-salt of Cpd. A Microparticles in Rats
after s.c.
Administration
The microparticles of Example 3A are suspended in a vehicle containing sodium
carboxy methylcelIulose, D-mannitol, Pluronics F68 (poloxamer 188, a
copolymer of
ethyleneoxide andpropylene oxide, BASF AG, Ludwigshafen, Germany) and water
for
injection. 200 microliters of these suspensions were injected subcutaneously
to the shaved
skin at the left dorsal side of 8 weeks old female virgin Wistar rats (body
weight
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approximately 220 g). In this way 1 mg dose (per animal) of the calcium -Cpd.A
microparticles (Example 3A) and 2 mg dose (per animal)are applied to a group
of 6 animals.
The thickness of the skin will be measured by a micro-caliper at the side of
injection and the
contra-lateral non-injected side. As reference a suspension of non-
encapsulated drug
substance is injected in a dose of 60 microgram. In addition, placebo
microparticles made
out of PLGA 50:50 are also injected as control.
It can be shown whether no irritation is caused by the microparticle
formulation
according to the invention.
Example 5: The Production of Implants with CO. A-Calcium salt
1.5 g of the micronized calcium salt of Cpd. A (1:2 salt) and 10.7 g of a PLGA
50:50
(IV 0.65 dL/g) are mixed thoroughly through cryo-milling in liquid nitrogen.
The resulting fine
powder is extruded at 90 C through a ram extruder with a speed of 5 mm/min.
The implants
of 1.5 mm diameter are cut at 2 cm length. The implants are placed in
applicators, sealed in
aluminum foil and finally sterilized by using gamma-irradiation with a dose of
30 kGy.
In the following Examples, X-ray powder diffraction patterns are measured on a
Bruker D8
Advanced Series 2 Diffractometer, Detector: PSD Vantec-1 with Cu Ka (1.54A)
radiation.
Parameters are as described in the Examples, respectively.
Example 6: Crystals of CO. A in free form (internal zwitterionic salt form):
The X-ray diffractogram of the zwitterionic salt of Cpd. A (which is
obtainable as in
Reference-Example 1) is obtained (elemental analysis C 27.9% (28.0), H 4.6%
(4.7), N 9.4%
(9.3%), P 20.5% (20.6%) (theoretical values in brackets) and yields the
following Peaks (see
also Fig. 1):
Table a. Powder X-Ray Diffraction Peaks for the crystalline form of the
zwitterions of
Cpd. A from Reference-Example 1:
deg 2A d-spacing (A) Relative intensity
10.5 8.39 Medium
13.1 6.74 Medium
14.7 6.03 Medium
17.2 5.14 Medium
23.5 3.78 High
25.2 3.54 High
34.4 2.60 Low
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Type: 2Th locked - Start: 2.000 - End: 40.030 - Step: 0.017 - Step time:
107.s - Temp.:
25 C (Room) - Time Started: 0 s - 2-Theta: 2.000 .
The melting point (m.p.) of the zwitterionic form is 237 C.
Example 7: Crystals of Cpd. A in Ca-salt (1:2) form:
a) Process for making the Crystalline Form of the 1:2 Calcium Salt of Cpd. A
(1
equivalent Ca : 2 equivalents Cpd. A):
In a 1000 mL 3-necked flask with mechanical stirrer, 3.5g of Cpd. A and 540 mL
water are
added. This mixture is heated to about 90 C until everything goes into
solution. To this clear
solution a solution of 667m g calcium chloride dihydrate in 10 mL water is
added. A white
precipitate appears and the mixture is then cooled to room temperature and
stirred over
night. The precipitated Ca salt is then isolated by filtration, washed with
cold water and dried
in a vacuum oven at 60 C over night. This the Ca salt of a composition which
corresponds
to the dihydrate, m.p. >230 C. The Ca-content of the salt is 5,8 % (theory 5.9
%).
Here the calcium salt of Cpd. A has a stoichiometry of one calcium and two
Cpd. A
molecules.
b) The X-ray diffractogram of Ca-salt of Cpd. A as obtainable under a) yields
the following
Peaks (see also Fig. 2):
Table b. Powder X-Ray Diffraction Peaks for the Crystalline Form of the 1:2
Calcium
Salt of Cpd. A
deg 29 d-spacing (A) Relative intensity
7.9 11.25 high
10.6 8.33 low
12.1 7.31 medium
25.7 3.46 medium
27.4 3.25 medium
29.2 3.05 low
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Type: 2Th locked - Start: 2.000 - End: 40.030 - Step: 0.017 - Step time:
107.s - Temp.:
25 C (Room) - Time Started: 0 s - 2-Theta: 2.000
Example 8: Crystals of CO. A in Zn-salt (1:2) form:
a) Process for making the Crystalline Form of the 1:2 Zinc Salt of Cpd. A
(1equivalent
Zn: 2 equivalents Cpd. A):
In a 1000 mL 3-necked flask with mechanical stirrer, 3.5g of Cpd. A and 540 mL
water are
added. This mixture is heated to about 90 C until everything goes into
solution. To this
clear solution a solution of 811 mg Zinc chloride in 10 mL water is added. A
white precipitate
appears and the mixture is then cooled to room temperature and stirred over
night. The
precipitated Zn salt is then isolated by filtration, washed with cold water
and dried in a
vacuum oven at 60 C over night. This the Zn salt with a composition
corresponding to a
dihydrate. m.p. >230 C. The Zn-content of the salt is about 9.0 % (theory 9.3
%).
Here the Zinc salt of Cpd. A has a stoichiometry of one Zinc and two Cpd. A
molecules.
b) The X-ray diffractogram of Zn-salt of Cpd. A as obtainable under a) yields
the following
Peaks (see also Fig. 3):
Table c. Powder X-Ray Diffraction Peaks for the Crystalline Form of the 1:2
Zinc Salt
of Cpd. A:
deg 2e d-s acin (A) Relative intensity
6.7 13.14 high
9.5 9.28 low
12.5 7.08 Medium
17.7 5.01 Low
27.3 3.26 Medium
Type: 2Th locked - Start: 2.000 - End: 40.030 - Step: 0.017 - Step time:
107.s - Temp.:
25 C (Room) - Time Started: 0 s - 2-Theta: 2.000 .
Example 9: Crystals of Cpd. A in Mg-salt (1:2) form:
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a) Process for making the Crystalline Form of the 1:2 Magnesium Salt of Cpd. A
(1 equivalent Mg: 2 equivalents Cpd. A):
In a 20mL vial with magnetic stirrer, 74.20mg of Cpd. A and 15 mL water are
added. This
mixture is heated to about 90 C until everything goes into solution. To this
clear solution a
solution of 11.8 mg Magnesium chloride in 24 mL water is added. A white
precipitate
appears and the mixture is then cooled to room temperature and stirred over
night. The
precipitated Mg salt is then isolated by centrifugation and dried in a vacuum
oven at 40 C
over night. This gives the Mg salt of a composition corresponding to a
dihydrate; m.p.
>230 C. The Mg-content of the salt is about 3.4 % (theory 3.7 %)
Here the Magnesium salt of Cpd. A has a stoichiometry of one Magnesium and two
Cpd. A
molecules.
b) The X-ray diffractograrn of the Mg-salt of Cpd. A as obtainable under a)
yields the
following Peaks (see also Fig. 4):
Table d. Powder X-Ray Diffraction Peaks for the Crystalline Form of the 1:2
Magnesium Salt of BCpd. A
deg 29 d-s acin (A) Relative intensity
6.7 13.15 high
12.5 7.09 low
20.0 4.43 low
27.3 3.27 low
Type: 2Th alone - Start: 2.000 - End: 40.030 - Step: 0.017 - Step time:
0.3 s - Temp.: 25
C (Room) - Time Started: 0 s - 2-Theta: 2.000
